Combination of lactic acid bacteria and its use for the prevention and/or treatment of infections and inflammatory conditions

ABSTRACT

The disclosure is of a combination of lactic acid bacteria comprising: (a) a first component consisting of at least one strain of H 2 O 2 -producing lactic acid bacteria; and (b) a second component consisting of at least one strain of arginine-utilizing lactic acid bacteria. The disclosure is also of the use of this combination for making a food supplement, a hygiene product or a pharmaceutical preparation for the prevention and/or treatment of infections and inflammatory conditions caused by bacteria, viruses or fungi, especially in the mouth, vagina, urethra, nose, eyes and ears.

[0001] The present invention relates to a combination of lactic acidbacteria and its use for making a food supplement, a hygiene product ora pharmaceutical preparation for the prevention and/or treatment ofinfections and inflammatory conditions caused by bacteria, viruses orfungi, especially in the mouth, vagina, urethra, nose, eyes and ears.

[0002] Lactic acid bacteria are Gram-positive bacteria that producelactic acid by the fermentation of glucose. Streptococcus thermophilusis also included in this definition by convention.

[0003] It is well known that strains of lactic acid bacteria thatproduce H₂O₂ can act as regulators of the bacterial flora in bodyorifices and on mucous membranes. It has been demonstrated thatH₂O₂-producing lactic acid bacteria can antagonize E. coli, N.gonorrhoea, G. vaginalis, C. trachomatis, U. urealyticum and B. bivius.However, these bacteria are only of limited benefit when used in medicalpractice. This can be seen from the fact that preparations based onlactic acid bacteria (e.g. vaginal pessaries) intended for the treatmentof infections by the above microorganisms (e.g. vaginitis) are not heldin high regard by doctors, who prefer to treat their patients withantibiotics or chemotherapeutic agents.

[0004] To the best of the inventor's knowledge, no antibacterial orflora-regulating action in body orifices and on mucous membranes hasbeen attributed to arginine-utilizing lactic acid bacteria.

[0005] It has now been found surprisingly that the activity ofH₂O₂-producing lactic acid bacteria is considerably potentiated by theaddition of one or more strains of lactic acid bacteria that are capableof utilizing arginine. Arginine forms part of various small peptidesfound in biological fluids and it also occurs as free arginine. Manybacterial species utilize it for their own nutrition and growth.Arginine-utilizing lactic acid bacteria can therefore deprive other,pathogenic or potentially pathogenic bacteria of a certain quantity ofarginine, which —though not enough to terminate their growth—makes themmore susceptible to the action of the H₂O₂ produced by the lactic acidbacteria.

[0006] The present invention therefore provides a combination of lacticacid bacteria comprising:

[0007] (a) a first component consisting of at least one strain ofH₂O₂-producing lactic acid bacteria, and

[0008] (b) a second component consisting of at least one strain ofarginine-utilizing lactic acid bacteria.

[0009] The strain of lactic acid bacteria in component (a) is preferablychosen from a group made up of the strains of the species Lactobacilluscrispatus, Lactobacillus salivarius and Lactobacillus casei, while thestrain of lactic acid bacteria in component (b) is chosen from a groupmade up of the strains of the species Lactobacillus brevis,Lactobacillus gasseri and Lactobacillus fermentum. More especially, thestrain of lactic acid bacteria in component (b) is the Lactobacillusbrevis CD2 strain deposited in the DSM-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, on Feb. 6,1998 with access number DSM 11988 under the Budapest Treaty, or mutantsor derivatives thereof.

[0010] The ratio of the number of bacteria in component (a) to thenumber of bacteria in component (b) is preferably from 1:100 to 100:1,and more especially from 1:5 to 5:1, the most preferred ratio being 1:1.

[0011] The combination can be administered in the unit dosage formcomprising from 1×102 to 5×10¹¹ bacteria of component (a) and from 1×10²to 5×10¹¹ bacteria of component (b), preferably 1×10⁹ bacteria ofcomponent (a) and 3×10⁹ bacteria of component (b).

[0012] The combination can also be administered in the form of tablets,sucking tablets, sweets, chewing gum, gelatin capsules, pessaries,suppositories and micro-enemas, as well as pellets, dental creams andgels, denture powders, mouthwashes, dentifrices, sprays, suspensions andointments.

[0013] According to another embodiment of the invention, the combinationadditionally comprises at least one other strain of lactic acid bacteriachosen from a group made up of: Lactobacillus acidophilus, Lactobacillusbuchneri, Lactobacillus casei, Lactobacillus catenaforme, Lactobacilluscellobiosus, Lactobacillus crispatus, Lactobacillus curvatus,Lactobacillus delbrueckii, Lactobacillus jensenii, Lactobacillusleichmannii, Lactobacillus minutus, Lactobacillus plantarum,Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus gasseri,Lactobacillus fermentum, Bifidobacterium adolescentis, Bifidobacteriumangulatum, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacteriumeriksonii, Bifidobacterium infantis, Bifidobacterium longum,Bifidobacterium plantarum and Streptococcus thermophilus.

[0014] The combination can also comprise vitamins, quaternary ammoniumbases, mineral salts, antioxidants and anti-plaque agents.

[0015] The invention also relates to the use of a combination of lacticacid bacteria comprising:

[0016] (a) a first component consisting of at least one strain ofH₂O₂-producing lactic acid bacteria and

[0017] (b) a second component consisting of at least one strain ofarginine-utilizing lactic acid bacteria, for making a food supplement, ahygiene product or a pharmaceutical preparation for the preventionand/or treatment of infections and inflammatory conditions caused bybacteria, viruses or fungi, especially in the mouth, vagina, urethra,nose, eyes and ears. These infections and inflammatory conditionsinclude gingivitis, periodontitis, mucositis and stomatitis caused bydrugs and/or physical agents, Behcet's syndrome, diakeratosis of theoral cavity, glossitis, sore throat, sialadenitis, sialolithiasis,pemphigus, Lichen planus, Sjögren's syndrome, vaginosis, vaginitis,urethritis, prostatitis, proctitis, otitis, conjunctivitis, rhinitis,sinusitis, leucoplakia, aphthae, herpes, and infections withHelicobacter pilori in the oral cavity.

[0018] The combination can also be used to advantage for the treatmentof the oral cavity as an oral deodorant, antiinflammatory, anti-cariesand/or anti-plaque agent.

[0019] The following examples serve to illustrate the various aspects ofthe invention in more detail but should not be construed as in any waylimiting the invention.

EXAMPLE 1

[0020] The inhibitory effect of: H₂O₂-producing lactic acid bacteria(Component A) ; arginine-utilizing lactic acid bacteria (Component B) ;and the combination of the two strains, specifically in the ratio 1:1(Combination AB); on the growth of potentially pathogenic bacteria wasevaluated.

[0021] Briefly, the culture of lactic acid bacteria to be tested wasfirst adjusted to a neutral pH, because an acidic pH itself inhibitsbacterial growth. The suspension was subjected to sterile filtration,and the filtrate was used to impregnate a number of discs of absorbentpaper (30 μl of filtrate per disc). The discs were placed on a plate ofselective growth medium that had been inoculated with 0.1 μl ofGardnerella vaginalis (a strain which causes vaginosis and which wasisolated in a laboratory) together with a control disc that wasimpregnated only with 30 μl of distilled water. After incubation for 24h at 37° C., the inhibition of the growth of the pathogens was evaluatedby measuring the diameter of the halo around the disc in millimetres.

[0022] A second series of tests was carried out with Streptococcusmutans as the target pathogen, this species being the causative agent ofdental plaque and caries.

[0023] The characterization of the bacteria as H₂0₂ producers was doneby a classical benzidine peroxidase reaction, which revealsH₂0₂-producing colonies of bacteria by a blue coloration. The activityof arginine dehydrolase was determined to evaluate the ability of thelactic acid bacteria to utilize arginine (M.C. Manca de Nadra,Milchwissenschaft, 37 (1982) pp. 669-670].

[0024] The lactic acid bacteria were obtained from the American TypeCulture Collection (ATCC), Rockville, USA. Halo of inhibition (anti-G.vaginalis Bacterial strain activity, mm) H₂O₂ producer (Component A)  21Lactobacillus crispatus (ATCC 39197)  75 Lactobacillus salivarius (ATCC11741)  60 Lactobacillus crispatus +  63 Lactobacillus salivariusArginine utilizer (Component B) Lactobacillus brevis (ATCC 14869)  0Lactobacillus fermentum (ATCC 14931)  2 Lactobacillus brevis +Lactobacillus  0 fermentum Combination AB (ratio of A to B 1:1)Lactobacillus crispatus + 112 Lactobacillus brevis Lactobacilluscrispatus + 100 Lactobacillus fermentum Lactobacillus salivarius + 117Lactobacillus brevis Lactobacillus salivarius + 104 Lactobacillusfermentum Halo of inhibition (anti-S. mutans Bacterial strain activity,mm) H₂O₂ producer (Component A)  21 Lactobacillus crispatus (ATCC 39197) 98 Lactobacillus salivarius (ATCC 11741) 102 Lactobacillus crispatus + 99 Lactobacillus saiivarius Arginine utillzer (Component B)Lactobacillus brevis (ATCC 14869)  0 Lactobacillus fermenturn (ATCC14931)  1 Lactobacillus brevis + Lactobacilius  1 fermentum CombinationAB (ratio of A to B 1:1) Lactobacillus crispatus + 118 Lactobacillusbrevis Lactobacilius crispatus + 126 Lactobacillus fermentumLactobacillus salivarius + 121 Lactobacillus brevis Lactobacillussalivarius + 120 Lactobacillus fermentum

EXAMPLE 2

[0025] Sucking tablets with the following unit composition wereprepared: Combination AB (Lactobacillus salivarius + 4000 millionLactobacillus brevis, ratio 1:1) Mannitol 400 mg Saccharin 5 mgPolyoxyethylene 50 mg Mg stearate 15 mg Talc 25 mg Silica 5 mg

[0026] These tablets were administered to four volunteers who were toldnot to clean their teeth or use chewing gum during the previous week.The subjects took three tablets a day for one week, after meals,allowing the tablets to dissolve in their mouth. Clinical evaluationswere performed for both the dental plaque index and the gingival plaqueindex.

[0027] For the dental plaque the following scoring system was used onsix teeth (first upper molar on the right; upper central incisor on theleft; first upper premolar on the left; first lower molar on the left;lower central incisor on the right; first lower premolar on the right):

[0028] 0—no plaque at all

[0029] 1—no visible plaque

[0030] 2—visible plaque

[0031] 3—very obvious plaque.

[0032] The following scoring system was adopted for evaluating thegingival plaque, using the margin of the six teeth mentioned above:

[0033] 0—no inflammation

[0034] 1—slight inflammation

[0035] 2—moderate inflammation, with bleeding on contact

[0036] 3—marked inflammation, with a tendency to spontaneous bleeding.

[0037] The combined data obtained for the six teeth of the fourvolunteers were as follows: Dental plaque Gingival index plaque indexSubject 0 7 days 0 7 days No. 1 8 2 7 1 No. 2 9 3 10 3 No. 3 15 5 6 2No. 4 15 7 8 3

EXAMPLE 3

[0038] Four subjects with a clinical and histological diagnosis ofrecurrent aphthous ulcers were treated for ten days with six suckingtablets a day, whose composition is given in Example 2.

[0039] All the patients treated showed a complete cure of the ulcers atthe end of the ten-day course, and none of them had new ulcers duringthe following month.

[0040] To improve the flavour and appearance of the bacterialcombination AB, suitable colouring agents and sweeteners such assaccharin, mint oil and xylitol can be added, as is customary and wellknown to those skilled in the art.

[0041] The combination AB can be administered in the form of pellets,sweets, chewing gum, gelatin capsules, dental creams and gels, denturepowders, mouthwashes, dentifrices, tablets, pessaries, suppositories,sprays, suspensions and micro-enemas.

EXAMPLE 4

[0042] Preparation of a toothpaste Toothpaste base Percentagecomposition: Calcium phosphate dihydrate  37.5% Glycerol (85% in water) 30.0% Flavour (peppermint oil)  1.0% Sodium carboxymethylcellulose 1.0% Purified water  20.8% Sodium saccharin (1% aq. solution)  2.5%Sodium lauryl sulphate  2.0% Purified water  5.2% 100.0% Composition byweight: Calcium phosphate dihydrate 337.5 g Glycerol (85% in water)270.0 g Flavour (peppermint oil)  9.0 g Sodium carboxymethylcellulose 9.0 g Purified water 187.2 g Sodium saccharin (1% aq. solution)  22.5 gSodium lauryl sulphate  18.0 g Purified water  48.8 g 900.0 g

[0043] The glycerol was added to the calcium phosphate dihydrate, whichhad been ground and passed through a 50-mesh screen. The mixture wasallowed to undergo hydration, giving a dense homogeneous paste. Theflavour (1% of peppermint oil) was added at this point.

[0044] The sodium carboxymethylcellulose was hydrated in water overnight(concentration of 4.6% in purified water).

[0045] The solution of sodium saccharin was added to the resulting thickgel.

[0046] The polymer gel was poured into a mortar already containing thecalcium phosphate that had been hydrated with glycerol, and thecomponents were vigorously mixed.

[0047] An approximately 28% solution of sodium lauryl sulphate inpurified water was prepared separately.

[0048] The sodium lauryl sulphate solution was then added to the thickdicalcium phosphate paste.

[0049] The resulting thick homogeneous paste, which had good Theologicalproperties, was mixed for a few minutes and then refined by passing itthrough a refiner with rollers. This gave a snow-white homogeneous pastewith a pleasant mint aroma.

[0050] 38.5 g of lyophilized lactic acid bacteria (L. salivarius+L.brevis, 1:1; 10¹⁰ CFU/g) were passed through a 50-mesh screen and addedin small portions to the rest (770 g) of the above toothpaste base. Theresulting homogeneous paste had a slightly pale brown colour and a mintodour.

EXAMPLE 5 Preparation of Fast-Release Vaginal Tablets

[0051] Vaginal tablets coated with an effervescent layer were preparedby wet granulation. The tablets weighed 2100 mg each and contained 100mg of the combination of lactic acid bacteria as the active substance.

[0052] Each vaginal tablet had the following composition: Lyophilizedand screened lactic  100.0 mg acid bacteria (30 × 10⁹ of L. brevis, 30 ×10⁹ of L. salivarius and 90 × 10⁹ of L. plantarum per gram) Lactose1368.0 mg Cornstarch  246.0 mg Adipic acid  192.0 mg Sodium bicarbonate 150.0 mg Magnesium stearate  30.0 mg Stearic acid   9.0 mg Colloidalsilica   5.0 mg 2100.0 mg

1. Combination of lactic acid bacteria comprising. (a) a first componentconsisting of at least one strain of H₂O₂-producing lactic acidbacteria, and (b) a second component consisting of at least one strainof arginine-utilizing lactic acid bacteria, wherein component (a) isselected from the group consisting of the strains of the speciesLactobacillus crispatus, Lactobacillus salivarius and Lactobacilluscasei, and component (b) is selected from the group consisting of thestrains of the species Lactobacillus brevis, Lactobacillus gasseri andLactobacillus fermentum, provided that when component (a) isLactobacillus casei, component (b) is not Lactobacillus gasseri orLactobacillus fermentum and when component (a) is Lactobacilluscrispatus, component (b) is not Lactobacillus fermentum.
 2. Combinationaccording to claim 1, in which the strain of lactic acid bacteria incomponent (b) is the Lactobacillus brevis CD2 strain deposited in theDSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, Germany, on Feb. 6, 1998 with access number DSM 11988under the Budapest Treaty, or mutants or derivatives thereof. 3.Combination according to any one of claims 1-2, in which the ratio ofthe number of bacteria in component (a) to the number of bacteria incomponent (b) is from 1:100 to 100:1.
 4. Combination according to claim3, in which the said ratio is from 1:5 to 5:1.
 5. Combination accordingto claim 4, in which the said ratio is 1:1.
 6. Combination according toany one of the previous claims in unit dosage units comprising from1×10² to 5×10¹¹ bacteria of component (a) and from 1×10² to 5×10¹¹bacteria of component (b).
 7. Combination according to claim 6,comprising 1×10⁹ bacteria of component (a) and 3×10⁹ bacteria ofcomponent (b).
 8. Combination according to claim 6 or 7, in the form oftablets, sucking tablets, sweets, chewing gum, gelatin capsules,pessaries, suppositories and micro-enemas.
 9. Combination according toany one of claims 1-5, in the form of pellets, dental creams and gels,denture powders, mouthwashes, dentifrices, sprays, suspensions andointments.
 10. Combination according to any one of the previous claims,additionally comprising at least one other strain of lactic acidbacteria, chosen from a group made up of Lactobacillus acidophilus,Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaforme,Lactobacillus cellobiosus, Lactobacillus crispatus, Lactobacilluscurvatus, Lactobacillus delbrueckii, Lactobacillus jensenii,Lactobacillus leichmanniit, Lactobacillus minutus, Lactobacillusplantarum, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillusgasseri, Lactobacillus fermentum, Bifidobacterium adolescentis,Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacteriumbreve, Bifidobacterium catenulatum, Bifidobacterium dentium,Bifidobacterium eriksonii, Bifidobacterium infantis, Bifidobacteriumlongum, Bifidobacterium plantarum and Streptococcus thermophilus,provided that when component (a) is Lactobacillus casei,said otherstrain is not Lactobacillus gasseri or Lactobacillus fermentum and whencomponent (a) is Lactobacillus crispatus, said other strain is notLactobacillus fermentum.
 11. Combination according to any one of theprevious claims, additionally comprising vitamins, quaternary ammoniumbases, mineral salts, antioxidants and anti-plaque agents.
 12. Use of acombination of lactic acid bacteria comprising: (a) a first componentconsisting of at least one strain of H₂O₂-producing lactic acidbacteria; and (b) a second component consisting of at least one strainof arginine-utilizing lactic acid bacteria, wherein component (a) isselected from the group consisting of the strains of the speciesLactobacillus crispatus, Lactobacillus salivarius and Lactobacilluscasei, and component (b) is selected from the group consisting of thestrains of the species Lactobacillus brevis, Lactobacillus gasseri andLactobacillus fermentum, provided that when component (a) isLactobacillus casei, component (b) is not Lactobacillus gasseri orLactobacillus fermentum and when component (a) is Lactobacilluscrispatus, component (b) is not Lactobacillus fermentum, for making afood supplement, a hygiene product or a pharmaceutical preparation forthe prevention and/or treatment of infections and inflammatoryconditions caused by bacteria, viruses or fungi, especially in themouth, vagina, urethra, nose, eyes and ears.
 13. Use according to claim12, in which the infections and inflammatory conditions includegingivitis, periodontitis, mucositis and stomatitis caused by drugsand/or physical agents, Behget's syndrome, diakeratosis of the oralcavity, glossitis, sore throat, sialadenitis, sialolithiasis, pemphigus,Lichen planus, Sjögren's syndrome, vaginosis, vaginitis, urethritis,prostatitis, proctitis, otitis, conjunctivitis, rhinitis, sinusitis,leucoplakia, aphthae, herpes, and infections with Helicobacter pilori inthe oral cavity.
 14. Use according to claim 12 or 13 for the treatmentof the oral cavity as a deodorant, antiinflammatory, anti-caries and/oranti-plaque agent.
 15. The composition according to claim 1 or 2,wherein component (a) is Lactobacillus crispatus and component (b) isLactobacillus brevis.
 16. The composition according to claim 1 or 2,wherein component (a) is Lactobacillus salivarius and component (b) isLactobacillus brevis.
 17. The composition according to claim 1, whereincomponent (a) is Lactobacillus salivarius and component (b) isLactobacillus fermentum.
 18. The composition according to claims 15-17,wherein the ratio of the number of bacteria in component (a) to thenumber of bacteria in component (b) is 1:1.